Trypanosoma cruzi, a protozoan parasite, is the causative agent of Chagas' disease (American trypanosomiasis), an infection of cardiac muscle cells and nerve ganglia that affects over 20 million persons in South and Central America, with over 90 million at risk. For practical purposes, chronic Chagas' disease is incurable, and drugs used for early infections can produce adverse side effects. Preliminary results from our laboratory (Craik laboratory) suggests a parasite cysteine protease (cruzain) is essential for parasite replication and transformation between stages of the T.cruzi life cycle. Our laboratory has cloned and expressed cruzain. In collaboration with the Fletterick Laboratory, the structure of cruzain was determined. The structural information will enable the use of computer screening techniques (DOCK 3.0) for the identification of nonpeptide, small molecule inhibitors that may be potent competitive or irreversible inactivators of the protease and that could eventually be developed into drugs. As a next stage in the "rational drug design" strategy, we will combine synthetic chemistry with computer modeling to identify modifications of promising lead compounds which can enhance their use in vivo. In addition, we shall use the expression system for the mutational analysis of the enzyme to identify amino acids that play potential functional or structural roles. We will first use computer graphics (INSIGHT and MIDAS) to model the amino acid replacement in the known cruzain structure to build a hypothetical structure of the mutant enzyme. We will then proceed to mutagenize, express, purify and characterize the mutant protein. We expect our proposed work will not only be relevant to the study and treatment of Chagas' disease, but will be a model strategy by which to study and target a number of other protozoan parasites that express structurally and biochemically similar enzymes.